Conventionally, in developing new drugs, candidate drugs are screened by patch clamping or a method using a fluorescent pigment or a light emitting indicator, while using cell electrical activities as indexes.
In the patch clamping, ion transport via a single channel protein molecule in a small portion, a patch, of a cell membrane attached to a tip of a micropipette is electrically recorded with a microelectrode probe. This is one of a few methods that can be used to examine functions of a single protein molecule in real time (See, e.g. Molecular Biology of the Cell, Third Edition, Garland Publishing Inc., New York, 1994, Bruce Alberts et al., Japanese Version, Saibou no Bunshi Seibutugaku Dai San Pan pp. 181-182, 1995, Kyoikusha).
Alternatively, it is possible to employ the aforementioned method using a fluorescent pigment or a light emitting indicator which emits light in response to concentration changes in a specific ion so as to measure cell electrical activities while monitoring intracellular ion transport.
However, the patch clamping requires special techniques for preparation and manipulation of a micropipette and also much time for measuring one sample. Hence, the patch clamping is not suitable for screening many drug candidate compounds at high speed. On the other hand, the method using a fluorescent pigment or the like can screen many drug candidate compounds at high speed; however, it requires a process of staining cells, and pigments not only discolor the background, but also are decolorized with time during measurement, thus resulting in a inferior S/N ratio.
WO02/055653 discloses a conventional device for measuring extracellular potentials, including a substrate having a unit for holding cells, and electrodes on the unit. This device can provide the same high quality of data as those obtained by the patch clamping, and also can measure many samples at high speed and as easily as the method using a fluorescent pigment.
An operation of the conventional extracellular potential measuring device will be described in detail as follows with reference to accompanying drawings.
FIG. 45 shows a cross sectional view of conventional extracellular potential measuring device 49. Well 40 contains culture solution 48A, and examined cell 47 is captured and held in a cell holder provided on substrate 42. The cell holder consists of pocket 41 provided in substrate 42, and through-hole 44 linked with pocket 41 via opening 44A. Through-hole 44 contains measuring electrode 45 as a sensor for outputting a potential of culture solution 48B inside through-hole 44 via wiring.
During measurement, cell 47 to be examined is tightly held in opening 41A of pocket 41 by a suction pump from a side towards through-hole 44. Then, electric signal 49A generated by activities of cell 47 is detected by measuring electrode 45 provided inside through-hole 44 without leaking into culture solution 48A inside well 40.
In this conventional extracellular potential measuring device 49, through-hole 44 is formed at the deepest point of pocket 41. For this structure, even when cell 47 is held inside pocket 41, if a cell membrane adheres onto a portion pocket 41 other than opening 41A, culture solution 48B inside through-hole 44 is electrically conducted with culture solution 48A inside well 40, hence preventing high precision measurement.
It is also impossible to examine whether or not cell 47 is held in pocket 41 and whether or not the cell membrane adheres as to cover through-hole 44.
Substrate 42 has two openings 41A and 41B having different diameters in both sides thereof. Opening 41A of pocket 41 for holding cell 47 has a diameter ranging about from 10 to 30 μm, and opening 44B of through-hole 44 opening on substrate 42 has a diameter ranging from 1 to 5 μm. Accurate formation of this structure requires two masks. The first mask is used to form pocket 41 by dry etching by photolithography, and then the second mask is used to form through-hole 44 by dry etching by photolithography.
However, conventional device 49 requires time to manufacture inexpensively since the masks may be misaligned during the dry etching after the dry etching, and since the masks require separate dry etchings.
Conventional device 49 also requires at least one of a pressurizing of culture solution 48A from well 40 and a depressurizing of culture solution 48B inside through-hole 44 in order to keep cell 47 inside pocket 41. At this moment, culture solution 48B needs to be introduced by a pressure difference into pocket 41 so as to contact measuring electrode 45. The pressure difference may be determined to be an appropriate value, hence allowing culture solution 48B to form a meniscus shape at opening 44B of through-hole 44 stably.
At opening 44B having a straight line shape shown in FIG. 45, the appropriate pressure difference capable of forming a meniscus shape of culture solution 48B ranges in a narrow range. In other words, a slight deviation of the pressure difference from the optimum values breaks the meniscus shape, thereby failing to keep culture solution 48B in a constant quantity.